| Cotton bollworm, Helicoverpa armigera (Hiibner) is a lepidopteran polyphagous pest. This pest has developed various levels of resistance to many insecticides including organochlorines, organophosphates, carbamates and pyrethroids. Since 1997, Bt cotton has been commercialized and effectively suppressed H. armigera field populations in northern China. However, insecticide spray is still one of the main control methods for cotton pests, because the damage of mirid bugs (non-target insects of Bt cotton) becomes serious and the bollworms still cause damage for the lower expression of Bt protein in the late cotton season. A higher fenvalerate resistance of H. armigera from northern China than that before Bt cotton was detected in 2011. In the present study, resistance to three different insecticides, fenvalerate, phoxim and emamectin benzoate, was inspected and mechanisms of field-evolved resistance to fenvalerate were detected in diffenent field populations using three approaches (bioassay, biochemical assay, and molecular screen). Toxicity baselines of four new insecticides (emamectin benzoate, spinosad, chlorfenapyr and chlorantraniliprole) against field populations of H. armigera were established in order to provide basic data for resistance risk assessment. Laboratory selection of resistance to emamectin benzoate was further carried out to assess the ealized heritability and resistance risk.1. Resistance monitoring and resistance mechanisms to fenvalerate in filed populations of H. armigeraResistance to fenvalerate and phoxim was measured by the topical application bioassay in 18 field populations of H. armigera collected in 2013 from northern and northwestern China. All 18 field populations had high resistance to fenvalerate (RFs:256-to 1806-fold), low or middle resisitance to phoxim (RFs:1-to 12-fold). The LD50 values of emamectin benzoate in these populations were between 0.367-2.235 ng/larva, and most of them are less than the LD50 value of the susceptible strain (1.028 ng/larva).Mechanisms of fenvalerate resistance were detected in field populations using three approaches including bioassay, biochemical assay, and molecular screen.The mortality of the discriminating dose (DD) of fenvalerate (0.08 μg/larva) with or without PBO (cytochrome P450 oxidase inhibitor) or DEF (esterase inhibitor) was tested in 7 field populations from each of the 7 provinces. Under the DD of fenvalerate, survivals were between 96% and 100% in the 7 field populations. Under DD+DEF, survivals decreased by 6%-15%. Otherwise under DD+PBO, survivals decreased by 68%-94%. These synergism results show that oxidase-mediated detoxification is the major contribution to fenvalerate resistance, and esterase-mediated detoxification maybe involve in resistance to some extent.Detoxifying enzyme activities of the fourth instar larvae were measured. Compared with the susceptible SCD strain, the 7 field populations had increased P450 monoxygenase activities against three different substrates (p-nitroanisole, ethoxycoumarin and methoxyresorufin). P450-mediated MROD activities in the 7 field populations were 3 to 16-fold higher than that of the susceptible SCD strain, and MROD activities were correlated to resistance levels of fenvalerate. However, esterase acitivities were lower in the field populations than that in the SCD strain. This result of detoxifying enzyme activities was consistent with the bioassay results of PBO and DEF synergists.The 42-fold resistance to fenvalerate in an Australian strain of H. armigera is due to a unique P450 CYP337B3, which arose from unequal crossing-over between two parental P450 genes CYP337B1 and CYP337B2. CYP337B3 is capable of detoxifying fenvalerate, but the two parental P450 genes showed no detectable fenvalerate detoxification. To investigate the possible role of CYP337B3 on fenvalerate resistance in Chinese populations, the frequencies of homozygous individual for CYP337B3 were investigated in 11 field populations and 2 laboratory strains. In the spatial point of view, the 7 populations from northern China had extremely high individual frequency of homozygous CYP337B3 (92%-100%), while the susceptible SCD strain has no CYP337B3 gene. In the temporal point of view, the individual frequency of homozygous CYP337B3 was 71%-96% in the 3 field populations (SW2008, SW2011, SW2013, collected from Shawan of Xinjiang in 2008, 2011 and 2013 respectively). SW2008 and SW2011 had high proportion of CYP337B3 homozygote (71% and 79%), but had limited resistance to fenvalerate (no resistance and 10-fold resistance). The above results suggest that in the field populations from northern China, there may be additional P450s contributing to fenvalerate resistance besides CYP337B3.2. Resistance risk assessment for four new insecticides in H. armigeraInspection of susceptibility and establishment of baseline of field populations to new insecticides are the basic components of resistance risk assessment. Susceptibility variations of 10 field populations collected in 2012 and 3 laboratory strains of H. armigera to four new insecticides (emamectin benzoate, spinosad, chlorfenapyr, and chlorantraniliprole) were evaluated by the leaf-dipping bioassay. The variation in susceptibility to chlorfenapyr among the 10 field populations was 52-fold, with LC50S ranging from 0.206 to 10.588 mg/L. The variation to emamectin benzoate was 19-fold, with LC50S ranging from 0.182 to 3.406 μg/L. The variation in susceptibility to spinosad was 6-fold, with LC50S ranging from 1.728 to 9.011 mg/L. The variation in susceptibility to chlorantraniliprole was 6-fold, with LC50S ranging from 0.338 to 1.739 mg/L. The variation in susceptibility to the four different insecticides may reflect the different insecticides use history and (or) the different intrinsic susceptibility among these geographic populations.Realized heritability (h2) of resistance to emamectin benzoate was caculated based on 10 generations laboratory selection of the susceptible SCD strain by the topical application bioassay. A 5.5-fold increase of resistance to emamectin benzoate was obtained through 10 generations of successive selection. The realized heritability of resistance to emamectin benzoate in H. armigera was 0.104 by using threshold trait analysis. According to h2, a 10-fold increase in LC50 would be expected in 9 generations if 90% of the population was killed at each generation. Thus, there exists certain resistance risk for emamectin benzoate in H. armigera. The adaptive resistance management to this chemical must be desiged before its extensive application. |
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